1. Technical Field
The present invention relates to a method and reagents for a fluorescence polarization immunoassay procedure for determining the amount of barbiturate in fluids, especially biological fluids such as serum, plasma or urine, and to a method of making the reagents. More specifically, the invention relates to (1) reagents (tracers and antibodies, and a kit containing the same) for determining the amount of barbiturate in a sample; (2) immunogen compounds used to raise antibodies; (3) synthetic methods (for making the tracer and immunogen compounds); and (4) analytical methods for conducting the assay.
2. Background Art
Barbiturates are central nervous system depressants. Therapeutically, they are used as sedatives, hypnotics and anticonvulsants. Although the legal availability of barbiturates has declined, they are frequently abused sedative or hypnotic drugs and are commonly used to commit suicide.
The physiological absorption, action and toxicity of barbiturates vary widely and are dependent on the nature of the 5-substituted groups and imino-hydrogens. Approximately 35% of the barbiturate in blood is plasma protein bound. Barbiturates are distributed in various tissues and organs. Barbiturates are primarily metabolized in the liver and, with a few exceptions, are generally excreted in urine mainly as nonactive metabolites.
The most commonly abused barbiturates are the short to medium acting: secobarbital, pentobarbital, amobarbital, etc. These are widely used to reduce excitation states due to the use of stimulants. Tolerance to these drugs can develop from chronic use, and death may occur from either overdose or abrupt withdrawal of the drug.
In the past, barbiturate levels in urine have typically been measured by high performance liquid chromatography (HPLC), gas chromatography (GC), enzyme immunoassay (EIA), substrate-linked fluorescence immunoassay (SLFIA) and radioimmunoassay (RIA). These methods are reasonably specific for detecting drug levels; however, they are not without drawbacks. HPLC and GC methods require sample extraction procedures and the assay time is lengthy. Both EIA and SLFIA involve enzyme reactions and have the following disadvantages:
1) the reagents are relatively unstable; 2) any components in the biological samples which may influence the enzyme reaction in EIA or SLFIA (such as enzyme inhibitors or enzymes which catalyze similar reactions) will affect the assay results; and 3) EIA and SLFIA measure either absorbance or fluorescence, and any compounds in the biological samples which may affect absorbance or fluorescence (such as lipid, hemoglobin, bilirubin or other chromophores or fluorophores) will affect the accuracy of the results obtained from these assays. RIA reagents have the following shortcomings: 1) short shelf-life; 2) radiation hazards; and 3) problems associated with the storage and disposal of radioactive materials.
Typically, competitive binding immunoassay are used for measuring ligands in a test sample. (For purposes of this disclosure, a xe2x80x9cligandxe2x80x9d is a substance of biological interest to be determined quantitatively by a competitive binding immunoassay technique.) The ligands compete with a labeled reagent, or xe2x80x9cligand analog,xe2x80x9d or xe2x80x9ctracer,xe2x80x9d for a limited number of binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
Fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane-polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of molecular rotation.
Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane-polarized light, the emitted light remains highly polarized because the fluorophore is constrained by the antibody from rotating between time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane-polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. As a result, the light emitted from the unbound tracer molecules is depolarized.
A problem that heretofore has prevented the accurate determination of barbiturates and other xe2x80x9cdrugs of abusexe2x80x9d in urine by fluorescence polarization techniques is that of riboflavin interference. Riboflavin, or vitamin B2, is a common constituent of many foods and of commercially available vitamin supplements. Riboflavin is excreted primarily in the urine and has a fluorescence spectrum quite similar to that of fluorescein. As a result, the presence of riboflavin in even moderate amounts in urine samples creates an interference which can produce erroneous results. While ordinary consumption of riboflavin is unlikely to produce more than trace amounts of riboflavin in the urine, test results can readily be distorted by the consumption of excessive quantities of vitamin supplements by persons wishing to prevent detection of barbiturate use.
The present invention is characterized by a more uniform cross-reactivity for the commonly used barbiturates. Further, this invention offers an advance in the art, in that tracers, a method for making the tracers, and an assay using the tracers, are provided specifically for the determination of barbiturates without riboflavin interference.
The present invention is directed to a fluorescence polarization assay for barbiturates; to tracers, immunogens and antibodies for use in the assay; to a reagent kit; and to methods for making the tracers, immunogens and antibodies,
A first aspect of the invention relates to the discovery of unique tracers and immunogens having novel structures. According to the first aspect of the invention, the tracers and the immunogens are represented by the structural formulas shown in FIG. 27 and FIG. 2, respectively, wherein, for the tracers:
(1) W is oxygen or sulfur;
(2) R1 is an alkyl, alkenyl, aryl, or alkynyl group having a total of from 1 to 12 carbon atoms, and including from 0 to 1 halogen atoms, arranged in a straight or branched chain, and including up to one aliphatic or aromatic ring structure;
(3) R3 is R4Fl;
(4) Fl is fluorescein or a fluorescein derivative; and
(5) R4 is a linking group which:
(a) has a total of from 0 to 15 carbon atoms and heteroatoms, arranged in a straight or branched chain, and includes up to one aliphatic or aromatic ring structure, said heteroatoms being O, N, S, P or F; and
(b) has a total of from 0 to 5 heteroatoms;
and, for the immunogens:
(1) W is oxygen or sulfur;
(2) R1 is an alkyl, alkenyl, aryl, or alkynyl group having a total of from 1 to 12 carbon atoms, including from 0 to 1 halogen atoms, and including up to one aliphatic or aromatic ring structure;
(3) R2 is Rxe2x80x94Q;
(4) Q is a poly(amino acid), a poly(amino acid) derivative or another immunogenically active carrier; and
(5) R is a linking group which:
(a) has a xe2x80x94CH2xe2x80x94, a xe2x80x94CHxe2x95x90, a 
xe2x80x83or a xe2x80x94NHxe2x80x94 at that end of said linking group which is linked to Q;
(b) has a total of from 0 to 20 carbon atoms and heteroatoms, arranged in a straight or branched chain, and includes up to one aliphatic or aromatic ring structure, said heteroatoms being O, N, S, P or F; and
(c) has a total of from 0 to 7 heteroatoms.
A second aspect of the invention relates to monoclonal or polyclonal antibodies prepared against the novel immunogen. According to the second aspect of the invention, antibodies are prepared in response to a compound according to FIG. 2. The most preferred antibodies of the present invention are prepared in response to the novel immunogen shown in FIG. 29.
According to a third aspect of the invention, an immunogen may be synthesized by a method comprising the step of coupling a compound represented by the structural formula shown in FIG. 2 wherein:
W is oxygen or sulfur;
R1 is alkyl, alkenyl, aryl or alkynyl having a total of from 1 to 12 carbon atoms, arranged in a straight or branched chain, and containing up to one aliphatic or aromatic ring structure;
R2 is CH xe2x80x94Rxe2x80x94X;
X is NH2, Cl, Br, I, OH, CO2H, Oxe2x80x94Cxe2x80x94Cl, 
xe2x80x83xe2x80x94CHO, 
xe2x80x83or 
xe2x80x83and
R is a linking group including up to 7 heteroatoms and having a total of from 0 to 20 carbon atoms and heteroatoms arranged in a straight or branched chain and containing up to one aliphatic or aromatic ring structure;
with a poly(amino acid), a poly(amino acid) derivative or another immunologically active carrier.
According to a fourth aspect of the invention, a method is provided for making a tracer by coupling a compound represented by the structural formula shown in FIG. 27, wherein;
W is oxygen or sulfur;
R1 is alkyl, alkenyl or alkynyl having a total of from 1 to 12 carbon atoms arranged in a straight or branched chain and containing up to one aliphatic or aromatic ring structure;
R3 is CH2xe2x80x94Rxe2x80x94Y;
Y is xe2x80x94NH2, COOH, xe2x80x94COCl, SO3H; SO2Cl, SH, CHO, CN, OH, or I; and
R is a linking group including up to 10 heteroatoms, having a total of from 0 to 20 carbon atoms and heteroatoms, arranged in a straight or branched chain and containing up to one aliphatic or aromatic ring structure;
with fluorescein or a derivative of fluorescein.
Preferably, the tracer is prepared by coupling a precursor of the structural formula shown in FIG. 27 wherein:
W and R3 are as defined above;
R1 is alkyl having 4 or 5 carbon atoms arranged in a branched chain and containing no ring structure;
Y is NH2 or COOH; and
R is a linking group including up to 3 heteroatoms, having a total of from 3 to 5 carbon atoms and heteroatoms arranged in a straight or branched chain and containing no ring structure.
Preferred derivatives of fluorescein include amino, amido, amidino, urea, thiourea, carbamido, thiocarbamido or triazinylamino derivatives. Most preferred at the present time are the amino derivatives, particularly aminomethylfluorescein.
A fifth aspect of the invention relates to the elimination of potential fluorescence interference by riboflavin. Riboflavin binding protein (RBP) is added either directly to each sample or to one or more of the reagents utilized in the assay, wherein it binds all riboflavin present into RBP-riboflavin complexes, thus eliminating fluorescence interference. Other fluorescence-quenching substances may also be utilized for this purpose.
According to a sixth aspect of the invention, a process for detecting or measuring the concentration of barbiturates is provided. A sample is contacted with barbiturate antiserum, and a fluorescein-containing barbiturate derivative capable of producing a detectable fluorescence polarization response to the presence of the barbiturate antiserum. Plane-polarized light is then passed through the solution to obtain a fluorescence polarization response, and this response is detected as a measure of the amount of barbiturate in the sample.
A seventh aspect of the present invention relates to a stabilized reagent kit which is useful for detecting commonly-used barbiturates in a single assay. The reagent kit contains novel tracers, and salts thereof, which are useful as reagents in the novel method of the present invention. Other components of the reagent kit in accordance with the instant invention include a solution containing an amount of riboflavin binding protein which is effective to reduce fluorescence interference by riboflavin and an antibody reagent which has been raised against an immunogen which is capable of specifically recognizing and binding the commonly-used barbiturates and the novel tracer reagents of the present invention.
Further objects and attendant advantages of the invention will be best understood from a reading of the following detailed description taken together with the Figures and the Examples.